Adult hematopoiesis
Hematopoiesis is the physiological process which allows the production and continuous and regulated renewal of the whole of the formed elements of blood from a small contingent of cells called hematopoietic stem cells (HSCs). In human adults, hematopoiesis lies in bone marrow within specific localizations (called niches) where it is closely regulated by the means of secreted factors and/or direct interactions with the cell component and the extracellular matrix which surround it, which form the <<microenvironment>>. Deregulation of this process in many cases leads to the development of malignant hemopathies. Study of the regulation of hematopoiesis at different levels of its differentiation (HSCs, immature progenitors, committed progenitors, mature and functional cells) has given the possibility of emphasizing the importance of the microenvironment and of the signals which it generates. Indeed, it is the development of co-cultivation systems of hematopoietic cells on stromal medullary cells which, by mimicking a microenvironment, has given in vitro access to the study of myelopoiesis and lymphopoiesis B as well as of immature progenitors LTC-IC (Long Term Culture-Initiating Cells). In vitro lymphopoiesis T, as for it, has only been possible in the presence of a thymic microenvironment or directly in organ-typical cultures of the FTOC (Fetal Thymic Organ Culture) type. Its study on medullary stroma was recently made possible by modifying these stromas (overexpression of Delta like-1 by gene transfer) (De Smedt et al, Blood Cells Mol Dis, 2004, 33(3):227-32).
These data emphasize the importance of better understanding the signals emitted by the microenvironment for studying and analyzing normal or pathological hematopoiesis in its global component. This is all the more important since several recent studies conducted on mice suggest that perturbations of the hematopoietic microenvironment/of niches may lead to development of myeloproliferative syndromes (MPS). Also in humans, an increasing number of arguments are in favor of the role of the microenvironment in the pathogenesis of leukemias and MPSes by providing an environment favorable to the development of the pathological clone, to the detriment of normal hematopoiesis. This assumption is supported by the observation of development of MPSes in certain allografted patients, while the relevant donors do not develop any of them.
Medullary Niches with Hematopoietic Stem Cells
Although the concept of a hematopoietic niche was introduced for the first time in 1978 by Schofield, it is only from the beginning of the years 2000 that a set of experimental arguments contributes to demonstration of its existence.
Present knowledge of the hematopoietic microenvironment reports the existence of two niches regulating the HSCes and respectively called endosteal niche and vascular niche. The endosteal niche is located in contact with the bone and is made of osteoblasts, fibroblasts and adipocytes which all originate from the mesenchymatous stem cell (MSC), as well as of osteoclasts deriving from HSCs and involved in bone resorption. Very recent studies show that osteoblasts and adipocytes may derive from Nestin+ MSC, the quiescence, proliferation and differentiation of which would be regulated by the sympathetic nervous system (SNS), therefore suggesting the role of the central nervous system in the modeling of the niches, the regulation of the HSCs and their mobilization. The vascular niche, as for it, consists in a network of fenestrated vessels, consisting of endothelial cells, deriving from endothelial stem cells (ESCs). So-called <<CAR>> (CXCL12 Abundant Reticular) cells present in bone marrow, would also participate in the forming of these niches via the production of CXCL12/SDF-1.
HSCs maintain constant dialog with their niche, where they are regulated via direct contacts with different stromal cells as well as through microenvironmental factors (concentrations of Ca2+ and of O2). A certain number of ligands/receptors pairs such as angiopoietin-1/Tie2, Jagged/Notch, Frizzled/Wnt, Sonic Hedgehog/Patched, as well as adherence molecules of the VLA4, VLA5, CD44, N-cadherin type are involved in the interactions between the osteoblast and HSC, and would regulate the quiescence/self-renewal equilibrium of the latter. Further, the SP (Side Population) functionality for drug exclusion, one of the characteristics of the HSCs, may be intimately related to their localization within the niches and notably endosteal niches. The diffusible factors as for them are represented by certain hormones (parathyroid hormone, serotonin, . . . ) BMPs (bone morphogenic proteins), cytokines (SCF, VEGF, TPO . . . ) and chemokines (CXCL12/SDF-1, IL8 . . . ).
The endosteal niche, center of a relative hypoxia to the contact of the bone, would be involved in maintaining stem cells in a quiescent state, via interactions with osteocompetent cells of the mesenchymatous line (osteoblasts, adipocytes, fibroblasts) and of the hematopoietic line (osteoclasts and endothelial cells).
In addition to its structural role of support, the extracellular matrix (ECM) also operates as a key regulator in the proliferation/differentiation/survival of HSCs/HPs within the niches. Among the components of this matrix, structural proteins are found including collagens and elastin, specialized proteins such as fibronectin and laminin, and regulatory proteins like metalloproteases and proteoglycans. Proteoglycans form a family of proteins, the major benefit of which is borne by its chains of glycosaminoglycans (GAGs). These GAGs intervene in the bioavailability of growth factors and of chemokines and protect them from their proteolytic degradation.
At the present time, while the impact of the bone system on hematopoiesis begins to be better known, little work has studied the mutual role of hematopoiesis in the physiology of bone remodeling. The vascular niche, where the oxygen concentration is higher, would rather be involved in the proliferation and differentiation of HSCs, although recent work has shown the importance of endothelial cells in the self-renewal of HSCs. However, to this day, it has definitively not been established whether both of these niches are distinct, or if they contribute to a common niche.
Understanding the Regulation of Hematopoiesis and Testing the Pharmacotoxicology of Medicaments
The mechanisms involved in the interactions between HSCs and osteocompetent cells within their regulatory niches still remain poorly known, whether in physiology or pathology. The understanding of these mechanisms is in fact limited because of the absence of an in vitro tool enabling studying hematopoiesis in a global context in particular integrating its microenvironmental aspects.
On the other hand, the development of novel therapeutic molecules requires the development of tests suitable for evaluating their toxicity and their efficacy on hematopoiesis and potentially on bone remodeling. Such a tool therefore has a major benefit on cognitive, pharmacological and therapeutic levels, in particular in hematology (di Maggio et al, Biomaterials, 2011, 32(2):321-9). Indeed, presently, there does not exist any culture system or only very few, allowing the study of normal or pathological hematopoiesis in the context of its microenvironment: the <<niche>> component is either absent (liquid culture in cytokine conditions), or restricted and poorly adapted (co-cultivation on stromal lines) or non-accessible (in vivo experimentation, graft). Among the rare studies reported to this day, the developed models are limited to interactions between certain stromal cells and HSCs, and they do not take into account the niche in its globality (de Barros et al, PLoS One, 2010, 8; 5(2)).
Therefore there is a major benefit in modeling a two-dimensional (2D) or three-dimensional (3D) niche in order to have an in vitro tool accessible and adjustable for studying hem atopoiesis in a context closer to physiology.
The object of the present invention is therefore to propose a 2D or 3D model of a hematopoietic niche, recreating the complexity of the medullary environment.
The invention consists of performing cellularization of biological (decellularized bone fragments) or manufactured supports, of variable calcium richness, with different types of stromal cells (osteoblasts, osteoclasts, adipocytes, mesenchymatous cells, endothelial cells) and of co-cultivating, on these cellularized supports, stem cells or hematopoietic progenitors (SP, ALDHstrong, CD34+, Lin− . . . ). Constituents of the extracellular matrix (GAG, fibronectin, collagens . . . ) are grafted on these supports in order to ensure good bioavailability of the factors produced by the stromal cells. In order to reproduce as close as possible the concentration variations of oxygen of the medullary microenvironment, the co-cultures are made at concentrations of O2 varying from 0.1% to 20%, preferentially from 1% to 3%.
This model enables revealing the SP potential of stem cells and could form an in vitro evaluation test of the nesting potential of a hematopoietic graft. It also allows study of the role of the cellular and humoral elements forming the hematopoietic niche on the cycling, proliferation, differentiation and mobilization of HSCs. This system will also allow study of the role of hematopoietic cells on osteocompetent cells and bone remodeling. Finally this model can be used as a pharmacological and toxicological tool for testing in vitro new drugs targeting hematological diseases and which may affect the cellular elements forming the hematopoietic niche. This model may therefore be used for pharmacological and toxicological studies of medicaments. This tool also allows the study of the role of hematopoietic niches in the deregulation of hematopoiesis characterizing certain malignant hemopathies.